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G Pohl

Publications and source records attributed to G Pohl.

At least 37 records · Page 2Linked to original sources
PubMed1986

Porcine tissue plasminogen activator. Immunoaffinity purification, structural properties and glycosylation pattern.

Tissue plasminogen activator was purified in high yield from pig heart by immunoaffinity chromatography and characterized by analysis of the glycosylation pattern and the N-terminal amino acid sequence. Comparisons with the human enzyme reveals residue exchanges in the A-chain at positions 3 (porcine Arg/human Gln) and 5 (Thr/Ile), and in the B-chain at positions 6 (Tyr/Phe), 10 (Thr/Ala) and 20 (Val/Ala). The glycosylation pattern for the porcine activator was determined by endoglycosidase treatment followed by gel electrophoresis. The A-chain contains a single high-mannose type of N-linked glycan structure and the B-chain contains a complex type of oligosaccharide. A similar but not identical pattern has been observed for the human activator, purified from melanoma cells.

Amino Acid Sequence↗
PubMed1986

Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator. Activations, inactivations and reactivations.

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50,000/52,000 and 17,000/20,000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420-421 and 423-424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420-421 and 423-424, convert the active one-chain activator into an 'inactive' zymogen, which is again 'activated' by plasmin cleavage.

Amino Acid Sequence↗
PubMed1985

Purification and some properties of carbonic anhydrase from bovine skeletal muscle.

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.

Amino Acid Sequence↗
PubMed1984

Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences.

Tissue plasminogen activator, separated into variants I and II (differing in Mr by 2000-3000), was reduced and [14C]carboxymethylated. Fragments from cleavages with enzymes and cyanogen bromide (CNBr) were separated by reverse-phase high-performance liquid chromatography and subjected to sequence degradations. All seven CNBr fragments were purified and found to be compatible with the cDNA-derived amino acid sequence [Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Ylverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., & Collen, D. (1983) Nature (London) 301, 214-221]. Chemical characterization of 93% of the 527 residues recovered in 50 peptides confirmed the indirectly deduced primary structure of the protein. The tryptic peptide patterns from the two variants were found to differ for one peptide (T15). Since carbohydrate was present in this peptide for variant I and since a marked difference in chromatographic behavior for T15 was observed in variant II, we conclude that carbohydrate differences in this peptide (i.e., Asn-184 in the numbering system of the cDNA-derived amino acid sequence) are the explanation for the size differences between variants I and II. Carbohydrate was also found at two other positions in the protein, corresponding to Asn-117 and Asn-448. However, a fourth potential glycosylation site, Asn-218, is apparently not utilized for carbohydrate attachment. The enzyme is inactivated by diisopropyl phosphorofluoridate, which covalently modifies the serine residue corresponding to position 478, identifying this as the active site serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Differences between uterine and melanoma forms of tissue plasminogen activator.

Tissue plasminogen activator purified from human uterine tissue exhibits differences in N-terminal starting positions in relation to the melanoma cell plasminogen activator usually studied. A new starting position is compatible with an additional N-terminal processing apart from those already known. Like the melanoma activator, the uterine activator was found to yield protein chains starting at either of two positions. One of these was identical between uterine and melanoma activators, whereas the other was unique in each case. The most abundant starting position for the uterine preparation was at a valine residue, apparently from cleavage of a Gln-Val bond, and corresponding to Val-7 of the longest form of the melanoma activator chain.

Amino Acid Sequence↗
PubMed1983

Differential proteolysis and evidence for a residue exchange in tissue plasminogen activator suggest possible association between two types of protein microheterogeneity.

The N-terminal part of native one-chain tissue plasminogen activator from melanoma cells is not homogeneous. The protein chain starts at two different positions, in all probability representing a processing difference in the N-terminus. Both 'long' L-chains and 3-residue shorter S-chains are present in the preparations. In addition, results compatible with a positional Ser/Gly microheterogeneity were obtained at a single position (position L-4 which is equal to S-1). The N-terminal tripeptide difference seems to be coupled to the possible microheterogeneity: L-chains contain Ser in this position, while S-chains appear to contain predominantly Gly.

Amino Acid Sequence↗
PubMed1983

Purification and characterization of a melanoma cell plasminogen activator.

The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. The cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three-step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine-Sepharose and Sephadex G-150. All solvents contained Tween-80 (0.01%) and, except for the last step, aprotinin. The final product had a specific activity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single-chain molecules. Evidence was obtained that four different types of activator variants occur. First and known previously, the one-chain form can be proteolytically cleaved into a two-chain form. Secondly, both the one-chain and two-chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one-chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N-terminal amino acids. Finally, a possible positional microheterogeneity was detected. Digestion with plasmin yields the two-chain form with disulfide-bonded polypeptide chains, 'A' and 'B' (from the N-terminal and C-terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains the active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one-chain form. A speculative explanation for this activity may possibly be the presence of an epsilon-amino group of a lysine residue at a position close to the bond cleaved in the two-chain form.

Amino Acid Sequence↗
PubMed1977

[Concordant medullary thyroid carcinoma in identical twins with discordant phaeochromocytoma (Sipple syndrome) (author's transl)].

The first case of Sipple syndrome in identical twins is reported. It is concluded that the concordant medullary carcinoma of the thyroid is the result of a germinal mutation, whilst the discordant solitary phaeochromocytoma is caused by a somatic mutation according to the two mutation model. The practical conclusion to be drawn from this report is that if one identical twin develops carcinoma of the thyroid or a phaeochromocytoma, the other twin should be throughly investigated and surgical exploration undertaken, even in the absence of clinical evidence of disease.

Adult↗